Dog heartworm disease and, to a lesser extent, cat heartworm disease due to infection by Dirofiliaria immitis is a significant problem in the United States and world-wide. (See Haddock (1987), "Canine Heartworm Disease: A Review and Pilot Study," Soc. Sci. Med., 24(3): 225-246; Knight (1987), "Heartworm Infection," Vet. Clin. North Am. Small Anim. Pract., 17(6); 1463-1518; Monchy et al. (1993), "Pulmonary Dirofilariasis," Med. Trop. (Mars.), 53(3); 366-371; Ro et al. (1989), "Pulmonary Dirofilariasis: The Great Imitator of Primary or Metastatic Lung Tumor. A Clinicopathologic Analysis of Seven Cases and a Review of the Literature," Hum. Path., 20(1); 69-76.) Infection begins when infective larvae (third stage--L3) from an infected mosquito are deposited on the skin of the animal. The infective larvae enter through the bite wound and burrow into the subcutaneous tissue. During the next two to four months, larvae will molt twice to become immature adult worms. The immature adult worms penetrate veins to enter the bloodstream and then migrate to the right side of the heart where they will continue to mature into adult worms. Once they become sexually active (generally about six to seven months post-infection), they will produce first stage microfilariae (i.e., L1 or active embryos) which will circulate in the bloodstream. The microfilariae require a mosquito host to further develop. While feeding, a mosquito will ingest microfilariae along with its blood meal. The microfilariae picked up by the mosquito change from the first stage to the infective third stage (L3) in about 10-30 days, depending on ambient temperatures, and then migrate to the mosquito's mouth parts where they can be injected into a new host animal, thereby completing the Dirofiliaria immitis' life cycle.
In infected animals, the adult worms (up to 10 to 12 inches in length) generally occur in the right heart chambers and associated vessels and can live up to six to seven years. Microfilariae (first stage larvae) can live in the bloodstream up to about three years. Visible symptoms (e.g., labored breathing, coughing, and unexplained exhaustion) from the disease are usually observed only in chronically infected dogs and cats, considerable damage to heart, lung, and kidney function can occur before symptoms are generally observed. Treatment (i.e., intravenous injections of an adulticide such as thiacetarsamide followed after about four to six weeks with a microfilaricide such as dithiazanine) can be effective (especially with early diagnosis of the infection). Such treatment, however, is generally expensive since hospitalization is recommended and, in some cases, forced rest for a prolonged period may be required. In some cases, the treatment may need to be repeated. Moreover, the treatment is not without risk to the animal since killed heartworms and/or microfilariae can result in blood clots which can lodge in, for example, the lungs. In many cases, especially in advanced cases where chronic symptoms are already evident at the time of diagnosis, the disease can be fatal even with treatment because of the increased risk of blood clots and other complications. In such advanced cases or where the animal cannot withstand the chemical treatment method, surgery may be necessary to remove the adult heartworms. Thus, prevention of the initial infection is a much preferred alternative since it is both safer and significantly less expensive. Although the risk of an initial infection can be significantly reduced by keeping pets indoors, this is not a viable alternative for many (and perhaps most) pet owners; moreover, infection can occur through the bite of a single infected mosquito. Administration of a prophylactic agent such as diethylcarbamazine on a daily basis or Ivermectin on a monthly basis to at-risk animals throughout the mosquito season (and several months thereafter) can be very effective in preventing the initial infection. Such preventive treatments should only be begun, however, once it is confirmed that no microfilariae are present in the blood since the treatment itself can cause serious problems, including death, in already infected animals. Even when using a preventive program, periodic testing for D. immitis is still recommended to insure the program is working properly and to detect any infection that may have been present, but not detected, before treatment began.
Ideally, all dogs and cats (both pets and strays) should be tested for the presence of D. immitis, preferably on a periodic basis. As noted above, such a test is especially important if the specific animal is to be placed on a regimen with a prophylactic agent to prevent infection. If D. immitis is detected in a given population, appropriate measures can be taken to eliminate the parasite in the affected animals and/or, if appropriate, to institute the appropriate preventive treatment in the yet-uninfected animals and otherwise limit the spread of the parasite.
Direct observation of microfilariae in the blood using a microscope can be used. The accuracy of this method, however, is substandard since adult heartworms may be present even in the apparent absence of microfilariae (e.g., only worms of one sex may be present; adult worms may be sexually immature or too old to reproduce; the concentration may be too low; or the animal's immune system may be successful in destroying the microfilariae). In addition, direct microscopic evaluation for mass screening of a large population of animals would be expensive due to the time and skill levels required for such individual evaluations. Currently the most accurate test available for screening blood samples for D. immitis in dogs is an antigen test. (This antigen test is not effective in cats.) Since this immunoassay appears to mainly detect the presence of the adult worm, it does not have the specificity and/or sensitivity required to detect infection by the parasite throughout its life cycle. This could be a problem if, for example, the antigen which is the analyte in this immunoassay were not found at a high level in all the larval stages and in the adult. Thus, there remains a need for an assay which is specific for D. immitis, is highly sensitive to the presence of the parasite in a host, and has the ability to detect D. immitis regardless of the stage in the life cycle the parasite may be in at the time of sampling. Moreover, there is a need for an assay which is relatively quick and easy to perform and which can, therefore, be used for general screening of the dog and/or cat population in a given location.
Currently there is no known assay that is useful for detecting the parasite in the mosquito vector. Nevertheless knowledge of the geographic extent and density of D. immitis in the vector population is an important factor in planning eradication programs. One must know where parasite-bearing mosquitoes are found, and veterinary authorities should be able to assess the geographic distribution of susceptible dogs and cats. Thus there is a clear need for an assay directed at the mosquito vector which is specific for D. immitis, and sensitive enough to detect the parasite in a single or small number of mosquitoes that may be harvested in the field. As in the case of an assay in samples from dogs and cats, an assay carried out on mosquito samples should be able to detect D immitis at any stage its life cycle within the mosquito.
The sensitivity and specificity of DNA-based assays in detecting bacterial, viral, and parasite DNA in clinical samples has been demonstrated. (See Riley et al. (1992), "Development of a Polymerase Chain Reaction-based Diagnosis of Trichomonas vaginalis," J. Clin. Microbiol., 30(2); 465-472; Wesley et al. (1997), "Application of Multiplex Polymerase Chain Reaction for Rapid Identification of Campylobacter jejuni and C. coli Associated with Reproductive Failure," Am. J. Vet. Res., 58(10); 1070-1075; Guadagnino et al., (1997), "Prevalence, Risk Factors, and Genotype Distribution, of Hepatitis C Virus Infection in the General Population; A Community-based Survey in Southern Italy," Hepatology, 26(4); 1006-1011; Kowalchuk et al. (1997), "Detection and Characterization of Fungal Infections of Ammophila arenaria (Marram Grass) Roots by Denaturing Gradient Gel Electrophoresis of Specifically Amplified 18S rDNA," Appl. Environ. Microbiol., 63(10); 3858-3865; Kuan (1997), "Detection and Rapid Differentiation of Human Enteroviruses Following Genomic Amplification," J. Clin. Microbiol., 35(10); 2598-2601.) There remains, however, a need for the development of a rapid, accurate, relatively inexpensive DNA-based assay for D. immitis in both host (i.e., dog and/or cat) and vector (i.e., mosquito) species. The present invention provides such a DNA-based assay.